Carotenoids are 40-carbon isoprenoid pigments synthesized by all plants, algae and cyanobacteria as well as by several non-photosynthetic bacteria and fungi. In plants, carotenoids are synthesized within plastids. The central pathway of isoprenoid biosynthesis in plastids begins with the production of isopentyl diphosphate (IPP), a C5 molecule which is the building block for all long-chain isoprenoids, from pyruvate and glyceraldehyde 3-phosphate in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Following isomerization of IPP to dimethylallyl pyrophosphate (DMAPP), three additional molecules of IPP are combined to yield the C20 molecule, geranylgeranyl pyrophosphate (GGPP). These 1′-4 condensation reactions are catalyzed by prenyl transferase-type enzymes GGPP synthases. There is evidence in plants that the same enzyme, GGPP synthase, carries out all the reactions from DMAPP to GGPP.
The polyene chain of carotenoids may extend from 3 to 15 conjugated double bonds, which are responsible for the carotenoid characteristic absorption spectra and confer specific photochemical properties. Due to these properties carotenoids are essential components in all photosynthetic organisms, where they fulfill indispensable functions in photosynthesis. Carotenoids also play a part in plant reproduction by furnishing flowers and fruits with distinct pigmentation designed to attract animals and enhance pollination and seed dispersion. Many of the orange, yellow, or red colors found in these organs are generated by accumulation of high concentration of carotenoids in the chromoplasts. In addition, degradation products of carotenoids comprise aromatic and flavoring compounds, and their presence in fruit appeal to animals.
In the last decade, carotenoid biosynthesis in plants has been described at the molecular level (reviewed in, e.g. Lu, S. and Li, L. 2008. J. Integr. Plant Biol. 50:778-785). The first committed step in the carotenoid pathway is the head to head condensation of two GGDP molecules to produce phytoene, the first C40 carotenoid, catalyzed by the enzyme phytoene synthase (PSY).
Insertion of conjugated double bonds (carotene desaturation) into the C40 chain of phytoene leads to the formation of visible polyene chromophore. The plant carotene desaturation is a membrane-bound complex reaction sequence. Four double bonds are introduced to phytoene by two enzymes, phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS), each catalyzing two symmetric desaturation steps to yield ζ-carotene and lycopene, respectively (FIG. 1). It was recently established that all intermediates in this part of the pathway are cis-configured and that a specific isomerase, CRTISO, operates in conjunction with ZDS to produce all-trans lycopene (Isaacson, T. et al. 2002. Plant Cell 14:333-342; Isaacson, T. et al. 2004. Plant Physiol. 136:4246-4255; Park, H. et al. 2002. Plant Cell 14:321-332). In addition, it has been predicted that other enzymes and cofactors are essential for this process, including a factor designated Z-ISO which is involved in 15-cis-ζ-carotene isomerization (Li F. et al. 2007. Plant Physiol. 144:1181-1189). Carotene desaturation is a redox reaction, linked to an extended redox chain, employing quinones as intermediate and molecular oxygen as a terminal electron acceptor. Molecular oxygen is reduced by means of a plastidic “terminal” (“alternative”) oxidase. Due to this complexity and the membrane-bound topology of the enzymes involved, a PDS-ZDS desaturation system has never been reconstructed in vitro with purified proteins. A paper of Chen Y. et al., published after the priority of the present invention describes the isolation and characterization of Z-ISO gene, and proposed that the encoded protein has a role in isomerization of the 15-cis-bond present in the PDS product, 9,15,9′-tri-cis-ζ-carotene to form the ZDS substrate 9,9′di-cis-ζ-carotene.
Cyclization of lycopene by either lycopene β-cyclase (Lcy-b) or lycopene epsilon-cyclase (Lcy-e), lead to β-carotene and α-carotene, respectively. Oxygenations of cyclic carotenes produce xanthophylls. In bacteria, a single phytoene desaturase enzyme, CrtI, carries out the phytoene to trans-lycopene conversion. Surprisingly, a transgenic CrtI desaturase is active in plants.
In plants, carotenoids are also precursors for growth regulators and developmental signals. The hormone abscisic acid (ABA) is produced from the xanthophylls violaxanthin and neoxanthin. It has been recently discovered that a cleavage derivative of β-carotene, possibly 13-apo-β-carotenone, serves as a graft-transmissible inhibitor of lateral shoot branching in Arabidopsis. 
There is growing interest worldwide in increasing the content of vitamins and other functional nutrients in crop plants (DellaPenna, D. 1999. Science 285:375-379; Lindsay, D. G. 2000. Trends in Food Sci. Technol. 11:145-151). Carotenoids play crucial role in determining quality parameters of fruits and vegetables (reviewed in van den Berg, H. et al. 2000. J. Sci. Food Agric. 80:880-912). All carotenoid species that contain β-ring can be converted to retinol and thus are precursors of vitamin A (pro-vitamin A). While this is the major importance of carotenoids in human nutrition, additional health benefits are attributed to their antioxidant activity in vivo (Stahl, W. and Sies, H. 2003. Mol. Aspects Med. 24:345-351). Consumption of xanthophylls (especially lutein) has been associated with prevention of age-related macular degeneration.
Epidemiological studies have associated carotenoids with reduces risk of cancer and other diseases in humans (Cooper, D. A. 2004. J Nutr. 134:221S-224S). Specific health benefits have been attributed to the carotenoids phytoene and phytofluene (Shaish, A. et al. 2008. Plant Foods Hum. Nutr. 63:83-86). Significant uptake of phytoene and phytofluene from tomato-based products was reported in humans (Aust, O. et al. 2005), and it was found that they are readily absorbed by normal and prostate tumor cells (Campbell, J. K. et al. 2007. Nutr. Res. 27:794-801). Phytoene was demonstrated in animal models as an effective sunscreen (Mathews-Roth, M. M. and Pathak, M. A. 1975. Photochem. Photobiol. 21:261-263).
Phytoene and phytofluene absorb light in the ultraviolet wavelength range. The absorption spectrum of phytoene is 276-297 nm (major peak 285-287 nm) and of phytofluene 331-367 (major peak 348 nm). Zeta-carotene absorption spectrum is 374-425 nm (major peak 395-400 nm). Therefore, these carotenoids may be used as sunscreen to protect the skin from damages inflicted by ultraviolet (UV) light. Indeed, protection of skin from UV light was demonstrated in humans fed with tomato-based foods or extracts (Stahl, W. et al. 2001. J. Nutr. 131:1449-1451), and this effect was attributed to phytoene and phytofluene (Aust, O. et al. 2005. Int. J. Vitam. Nutr. Res. 75:54-60). Based on these studies, it has been suggested that dietary carotenoids may contribute to life-long protection against harmful UV radiation (Stahl, W. et al. 2006. Photochem. Photobiol. Sci. 5:238-242). The potential of using phytoene and phytofluene as sunscreen is supported by previous studies that showed benefit in combining topical treatment with carotenoids in addition to oral supplementation (Palombo, P. et al. 2007. Skin Pharmacol. Physiol. 20:199-210). Accumulation of phytoene and to some extent of phytofluene has been reported upon the addition of the herbicide norflurazon (4-chloro-5(methylamino)-2-(3-(trifluoromethyl)phenyl)-3(2H)-pyridazinone) to the growth medium of the algae Dunaliella (Ben Amotz, A. et al. 1987 J. Phycol., 23:176-181). However, the use of such chemicals is possible only in culture-grown organisms.
The amount of phytoene and phytofluene, being the first ingredients in the carotenoid pathway, is relatively low in carotenoid-containing organisms. In view of the beneficial effects reported for phytoene and phytofluene, there is a recognized need for, and would be highly advantageous to have, carotenoid producing organisms having high amounts of phytoene and phytofluene.